PROJECT SUMMARY Induced pluripotent stem cells (iPSCs) are potential diagnostic and therapeutic tools for disease modeling and regenerative medicine, but remain severely limited by reprogramming induced senescence (RIS), which is degenerative changes within cellular progeny resulting in progressive loss of the mature phenotypes possessed by fully functional endogenous cells. RIS is a stress-induced, or premature senescence and is therefore potentially amenable to modulation via intervention in regulatory mechanisms. Several key pathways have been repeatedly implicated in triggering RIS, including cell cycle checkpoint regulators and mediators of autophagy, which is the process of degrading and recycling intracellular contents. Despite this knowledge, investigations aimed at harnessing these pathways to produce phenotypically stable and translatable, long-lived progeny from iPSCs for eventual diagnostic and therapeutic use have met limited success. Our lab demonstrated and published that RIS can be manipulated in iPSC-derived endothelial cells (iPSC-ECs), resulting in stabilization of phenotype and prolongation of function via overexpression of Sirtuin 1 (SIRT1), an NAD-dependent de-acetylase known to modulate mediators of senescence by downregulating the cell cycle regulator p53 and by inducing autophagy. Given this evidence, and in conjunction with additional supportive data, we hypothesize that direct attenuation of SIRT1 targets at critical time-dependent intervals during the reprogramming process will yield iPSC-ECs that display robust and stable endothelial-like phenotypes with direct translatable potential. In Aim 1 we will transiently suppress p53 and p16INK4a with targeted siRNA to knockdown their expression and will uncover the specific time-dependent windows during reprogramming to maximize endothelial function of iPSC-derived cells while circumventing RIS. In Aim 2 we will induce autophagy with the small molecule AMP kinase activators ML246 and Rg2 at critical time points during iPSC induction and EC-differentiation of iPSC-ECs to suppress the switch to RIS and promote longevity of iPSC-ECs and durability of their phenotype and function. The scientific aims reinforce the training goal, which is to develop the investigator?s career niche in vascular and stem cell biology and to develop tools that will form the foundation of an academic career as a vascular surgeon-scientist. The scientific goal of this project is to identify key time points and strategies to intervene during reprograming to overcome RIS in iPSC-ECs, and to generate phenotypically stable and long lived endothelial-like cells. Knowledge gained from this investigation will have broad applications to generate stable iPSC-derived cells for other organ systems beyond the vasculature alone. Durable and functional iPSC-derived cells, such as those that accurately recapitulate endothelial function, would be tools for personalized disease modeling for vascular disease and may be used for therapeutic intervention in regenerative or bioengineering applications such as cell therapy to help injured tissues repair or to replace tissues when damage is too far advanced.